FoxO3a Inhibits Tamoxifen-Resistant Breast Cancer Progression by Inducing Integrin α5 Expression

Simple Summary Tamoxifen is a mainstay of adjuvant treatment for estrogen receptor α-positive (ERα+) breast tumors, which account for over 70% of all the diagnosed breast malignancies. Unfortunately, ~30% of patients initially responding to the therapy will eventually relapse with an endocrine-resistant disease. Here, we show that the transcription factor FoxO3a can overcome tamoxifen resistance by inhibiting cell motility and invasiveness, the main features of tumor progression. The underlying mechanism could be ascribed to FoxO3a-dependent transcriptional up-regulation of the integrin α5 subunit of the α5β1 fibronectin receptor, a well-known membrane heterodimer controlling cell adhesion. Indeed, FoxO3a and integrin α5 expression is significantly correlated in ERα+ tumors, and FoxO3a protective effects are lost in cells with reduced levels of integrin α5. Therefore, a pharmacological increase in FoxO3a levels could represent an effective approach to be exploited in combination with tamoxifen treatment in order to restore the sensitivity to the therapy in refractory tumors. Abstract Resistance to endocrine therapy is still a major clinical challenge in the management of estrogen receptor α-positive (ERα+) breast cancer (BC). Here, the role of the Forkhead box class O (FoxO)3a transcription factor in tumor progression has been evaluated in tamoxifen-resistant BC cells (TamR), expressing lower levels of FoxO3a compared to sensitive ones. FoxO3a re-expression reduces TamR motility (wound-healing and transmigration assays) and invasiveness (matrigel transwell invasion assays) through the mRNA (qRT-PCR) and protein (Western blot) induction of the integrin α5 subunit of the α5β1 fibronectin receptor, a well-known membrane heterodimer controlling cell adhesion and signaling. The induction occurs through FoxO3a binding to a specific Forkhead responsive core sequence located on the integrin α5 promoter (cloning, luciferase, and ChIP assays). Moreover, FoxO3a failed to inhibit migration and invasion in integrin α5 silenced (siRNA) cells, demonstrating integrin α5 involvement in both processes. Finally, using large-scale gene expression data sets, a strong positive correlation between FoxO3a and integrin α5 in ERα+, but not in ER-negative (ERα−), BC patients emerged. Altogether, our data show how the oncosuppressor FoxO3a, by increasing the expression of its novel transcriptional target integrin α5, reverts the phenotype of endocrine-resistant BC toward a lower aggressiveness.


Generation of Tamoxifen-Resistant Cell Lines
The selection of tamoxifen-resistant MCF-7 (TamR), ZR-75 (ZR-75/TR), and T-47D (T-47D/TR) cell lines were obtained after long-term cultivation of parental cells in their own growing medium (GM), with increasing concentrations of tamoxifen (Tam), followed by chronic exposure to Tam 1 µM, as previously reported [9]. The acquired resistance was periodically verified by checking the lack of Tam-dependent growth inhibition compared to the parental cells.

Generation of FoxO3a Inducible Stable Cell Lines
TamR/TetOn-AAA cell line and the relative control (TamR/TetOn-V) were developed as previously described [9], using the Tet-On Gene Expression System (Clontech, Palo Alto, CA, USA). Briefly, TamR cells were first transfected with the regulator plasmid pTet-On, carrying the geneticin (G418) resistance gene, for the selection of successfully transfected cells. G418-resistant TamR/TetOn cells were, thus, isolated and further transfected with the pTRE-F3aAAA plasmid, bearing a cDNA encoding the entire open reading frame of a constitutively active form of the human FoxO3a gene. The zeocin resistance gene in the pTRE-F3aAAA plasmid assured proper selection of double transfected cells. Control cell lines (TamR/TetOn-V) were established following the same protocol but stably transfecting the pTRE backbone (vector only) in place of the F3aAAA cDNA insert. Pools of TamR/TetOn-AAA and TamR/TetOn-V cells were collected and cultured in TamR medium containing G418 and zeocin selection antibiotics, plus Tam 1 µM. Medium was replaced every 48 h. The tetracycline derivative doxycycline (Dox) (Sigma-Aldrich, Merck, Milan, Italy) was employed to induce F3aAAA expression.

siRNA-Mediated RNA Interference
In order to have effective depletion of FoxO3a (siF3a) and of integrin α5 (siα5) transcripts, validated stealth RNAi (Oligo ID: VHS41092 for FoxO3a and siRNA ID: s7549 for integrin α5) were used. Cells were grown in phenol-red free-GM (PRF-GM) without antibiotics for 24 h and then silenced in suspension either with siF3a or siScramble (150 pmol/dish), using lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific). Similarly, siα5 (100 pmol/60 mm dish) was employed for efficient integrin α5 knock-down. A Stealth RNAi TM siRNA (siScramble) lacking identity with known gene targets was used as a negative control. All siRNA were purchased from Invitrogen, Thermo Fisher Scientific Inc. After 8 h, medium was renewed, and silencing efficiency was monitored up to 72 h.

Growth Assay
Cells were plated in 12-well plates (8 × 10 4 cells/well) in PRF-GM. After 8 h, cells were synchronized overnight (ON) in PRF-serum-free medium (PRF-SFM) and then shifted in 2% PRF-charcoal-treated (PRF-CT) medium and treated or not with Tam 1 µM up to 72 h. Cells were collected, resuspended in 0.5% trypan blue solution, and counted through a Countess ® II Automated Cell Counter (Life Technologies, Thermo Fisher Scientific Inc., Monza, Italy).

Spheroid Assay
The 10 6 cells were plated in PRF-GM in 100 mm dishes, coated with 2% agar solution in sterile PBS. Cells were monitored until spheroids formation (72 h). Images were taken at 10× magnification using an Olympus dp50 camera and ViewFinder software. Spheroids with diameters ≥ 50 µm were counted and measured using ImageJ software. Data were represented as mean ± SD of three fields per condition.

Colony-Forming Assay
Cells were plated (10 3 /well) in duplicate in 6-well plates in GM and treated or not with Tam 1 µM, where needed. Medium and treatment were renewed every 2 days. After 10 days, surviving colonies were fixed, stained with crystal violet, and scanned. Colonies were counted using ImageJ software 1.52t and expressed as a percentage of surviving colonies.

Wound-Healing Scratch Assay
Cells were plated (6 × 10 5 /well) in GM in 6-well plates. After 3 days, confluence was reached, and a scratch was made. After 24, 48, and 72 h, cells were fixed and stained with Coomassie brilliant blue. Pictures were taken at 10× magnification using phase-contrast microscopy, and the percentage of wound closure was analyzed by using ImageJ software, quantifying the wound opening area.
For invasion experiments, the inner membrane of Boyden chambers was coated with 30 µL of Matrigel™ Basement Membrane Matrix (BD Biosciences, Becton Dickinson Italia S.p.A. Milan, Italy) (1:3 in PRF-SFM) and left drying at RT for 30 . The bottom of the well was filled with PRF-GM as for migration. Cells have been resuspended in PRF-CT and plated (10 5 cells/insert) on the Matrigel layer. After 72 h, invading cells were fixed, stained, and counted as in migration experiments.
2.11. RNA Extraction, Reverse Transcription, and Real-Time (RT)-PCR Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific Inc.), and 2 µg were reverse transcribed with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc.) according to the manufacturer instructions. cDNA was diluted with SYBR green Universal PCR Master Mix (Bio-Rad, Milan, Italy) and evaluated in triplicates by RT-PCR in an iCycler iQ (Bio-Rad, Milan, Italy). The following primers were used: FOXO3 forward 5 -CAAACCCAGGGCGCTCTT-3 and reverse 5 -CTCACTCAAGCCCATGTTGCT-3 ; ITGA5 forward 5 -GGCAGGACGCTATGTCC-3 and reverse 5'AGGGAAGGAGGTGTGTGACTC-3 . Additional samples lacking cDNA and primers served as negative controls. Each sample was normalized on its 18S rRNA content.
Relative gene expression levels were calculated on the basis of the untreated sample, and the results were reported as n-fold differences in gene expression.

Cloning and Luciferase Assay
The luciferase expression vector pGL3-human integrin α5 (pGL3pα5) encoding the 5flanking region of the human integrin α5 gene (ITGA5; promoter sequence NCBI accession ID: U48214), already described in a previous report from our group [27], was employed in luciferase assays. An almost canonical Forkhead responsive core sequence (FHRE) GTTTAC (nt-329/-323 from the ATG starting site) was identified using Gene Snap software on the published integrin α5 promoter sequence.
To investigate α5 transcriptional activation by FoxO3a, 7 × 10 4 cells were seeded on 24-well plates and transfected either with pGL3pα5 or pGL3pα5-del and the Renilla reniformis luciferase expression vector pRL-Tk (Promega, USA), using FuGENE HD. Luciferase activity was assessed by means of the Dual-Luciferase ® Reporter Assay System (Promega, USA).

cBioPortal Database
cBioPortal (https://www.cbioportal.org, last access 2 October 2021), a large-scale gene expression public data set from The Cancer Genome Atlas (TCGA) database, has been used to evaluate in silico the potential co-expression of FOXO3 and ITGA5 genes in BC patients cohorts [28]. The search was conducted over three BC mRNAs data sets:

Kaplan-Meier Analysis
Kaplan-Meier (K-M) analysis, based on the most updated version of a public available microarray database from breast cancer patients (http://kmplot.com/analysis/index.php? p=service&cancer=breast, last access 20 December 2021), was used to evaluate the prognostic value of FoxO3a and integrin α5 in breast cancer. Distant metastasis-free survival (DMFS) and relapse-free survival (RFS) were calculated in a cohort of luminal A patients treated with tamoxifen (DMFS = 485 and RFS = 577 patients), regardless of the chemotherapy treatment. Search parameters: Gene: FoxO3a and ITGA5; Split patients by: auto-select best cutoff; Censored at threshold, selected; Use only JetSet best probe set, selected; Quality control, remove redundant samples: checked; Array quality control: exclude biased arrays selected; Restriction: luminal A; Intrinsic subtype, selected as indicated. The p-values were calculated using the log-rank test and plotted in R.

Statistical Analysis
Statistical analysis was performed using Student's t-test and ordinary ANOVA test through the GraphPad Prism software. All data are reported as the mean ± standard deviations (s.d.) of at least three different experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. control.

FoxO3a Re-Expression Reduces the Aggressiveness of Tamoxifen-Resistant BCCs
The acquisition of a tamoxifen-resistant phenotype has been associated with a more aggressive behavior than that of responsive tumors, exhibiting a higher proliferation rate as well as an increased migrating and invading potential in response to activated cytokines/growth factors signaling cascades [29,30]. These features have been evaluated in our TamR BCC model and compared to the originating MCF-7 cell line [9]. As expected, TamR cells do not respond to tamoxifen treatment and grow more than the parental MCF-7, not only in adhesion ( Figure 1a) but also in suspension (Figure 1b), and are able to form much more and bigger colonies starting from single cells (Figure 1c). They are also much faster in closing the scratch during wound-healing assays, reaching almost the complete sealing of the gap after 72 h ( Figure 1d). Transmigration experiments (Figure 1e) confirmed that the wound closure is due to increased motility and not to the higher proliferation rate of TamR cells compared to parental MCF-7 cells. Finally, TamR cells also show an increased ability in invading an artificial basement membrane in invasion assays ( Figure 1f). A similar behavior was observed in two additional tamoxifen-resistant cell lines T-47D/TR and ZR-75/TR (Appendix A, Figure A1a-d).
As mentioned, we previously reported that FoxO3a is significantly downregulated in TamR cells, both at the mRNA ( Figure 2a) and protein (Figure 2b) level and that its re-expression is able to restore the antiproliferative response to tamoxifen in resistant cells (Figure 2c) [9]. Here, we show that FoxO3a is also a crucial modulator of processes involved in the progression of tumors that are refractory to antiestrogens. Indeed, FoxO3a overexpression in TamR cells strongly reduces their ability to grow in suspension and form spheroids (Figure 2d  As mentioned, we previously reported that FoxO3a is significantly downregulated in TamR cells, both at the mRNA ( Figure 2a) and protein ( Figure 2b) level and that its reexpression is able to restore the antiproliferative response to tamoxifen in resistant cells ( Figure 2c) [9]. Here, we show that FoxO3a is also a crucial modulator of processes involved in the progression of tumors that are refractory to antiestrogens. Indeed, FoxO3a overexpression in TamR cells strongly reduces their ability to grow in suspension and form spheroids (Figure 2d Similar results were obtained in an MCF-7-derived, Dox-inducible TamR/FoxO3a stable cell line (TamR/TetOn-AAA) when compared to the corresponding control cell line (TamR/TetOn-V), both developed as previously reported [9] and briefly described in the Section 2 section (Appendix A, Figure A1e-k). Thus, both the transient and the inducible overexpression of FoxO3a lead to comparable effects, and that no adaptive behaviors, potentially misleading, did occur in the stable cell lines. For this reason, the two Similar results were obtained in an MCF-7-derived, Dox-inducible TamR/FoxO3a stable cell line (TamR/TetOn-AAA) when compared to the corresponding control cell line (TamR/TetOn-V), both developed as previously reported [9] and briefly described in the Section 2 section (Appendix A, Figure A1e-k). Thus, both the transient and the inducible overexpression of FoxO3a lead to comparable effects, and that no adaptive behaviors, potentially misleading, did occur in the stable cell lines. For this reason, the two experimental approaches to overexpress FoxO3a have been used indistinctly throughout the work.

FoxO3a Modulates the Expression of the Integrin α5 Subunit of the Fibronectin Receptor
Integrins are transmembrane receptors involved in cell adhesion to the extracellular matrix (ECM), which have been involved, both positively and negatively, in tumor initiation and progression. In this context, we and others previously reported that the integrin α5 subunit of the fibronectin receptor has a protective role in BC, behaving as an anti-metastatic factor [31], at least in ERα+ tumors [27].
Accordingly, integrin α5 levels, but not β1, resulted in downregulated in TamR cells (Figure 3a). This might be due to increased fibronectin-driven ubiquitination and subsequent lysosomal degradation of the α5 subunit [32] in TamR cells, which express higher levels of fibronectin [33]. Lower α5 integrin levels were also observed in two additional tamoxifen-resistant cell lines, T-47D/TR and ZR-75/TR (Appendix B, Figure A2a and b, respectively). Therefore, we questioned if integrin α5 expression might be regulated by FoxO3a, having the transcription factor a similar trend of expression in TamR cells (Figure 2b). Indeed, FoxO3a overexpression (Figure 3b,c) was able to induce integrin α5, both at the mRNA ( Figure 3b) and protein (Figure 3c) level. A similar integrin α5 induction was observed in vivo in FoxO3a overexpressing TamR/TetOn-AAA-derived tumors, explanted from Dox-treated mice, compared to control TamR/TetOn-V tumors (Figure 3d) [9]. Again, no significant difference was observed in β1 levels in the same tissue extracts (Appendix B, Figure A2c).

PEER REVIEW
10 of 23  derived tumors, explanted from Dox-treated mice, and from TamR/TetOn-V control tumors [9] were used to evaluate FoxO3a and integrin α5 expression. β-Actin was used as a loading control.
(e) TamR were seeded and, the day after, they were starved for 24 h, pre-treated with alpelisib 1 μM for 30′ and then treated with IGF 10 ng/mL for 15′. Cytosolic proteins were used to detect phosphorylated proteins. β-actin was employed as a loading control. (f) TamR were seeded and treated as in (e). Cytosolic and nuclear extracts were subjected to WB analysis for the detection of  [9] were used to evaluate FoxO3a and integrin α5 expression. β-Actin was used as a loading control. (e) TamR were seeded and, the day after, they were starved for 24 h, pre-treated with alpelisib 1 µM for 30 and then treated with IGF 10 ng/mL for 15 . Cytosolic proteins were used to detect phosphorylated proteins. β-actin was employed as a loading control. (f) TamR were seeded and treated as in (e). Cytosolic and nuclear extracts were subjected to WB analysis for the detection of indicated proteins. Lamin B1 and GAPDH were used as loading controls. (g) TamR were seeded, and the day after, they were starved for 8 h. Of note, the PI3K inhibitor alpelisib, that has been recently approved in combination with fulvestrant for locally advanced or metastatic BC, by inhibiting the phosphorylation of PI3K downstream targets AKT and FoxO3a (Figure 3e) and, consequently, preserving FoxO3a nuclear sequestration, thus activation, even under GFs (IGF-1) stimulation Figure 3f), was able to slightly increase integrin α5 protein expression as soon as after 18 h incubation Figure 3g).
Finally, FoxO3a silencing (siF3a) led to an evident downregulation of the integrin α5 subunit, both in parental and TamR cells (Figure 3h), confirming that integrin α5 expression is regulated, at least in part, by this nuclear transcription factor.

FoxO3a and Integrin α5 Are Positively Correlated in ERα+ BC
FoxO3a-induced up-regulation of integrin α5 is not limited to TamR cells, but it also occurs in parental MCF-7 (Figure 3b,c), in the TamR-derived Tet-inducible stable cell line (Appendix B, Figure A2d,e) and in other ERα+ BC cell lines, T-47D and ZR-75, including their Tam-resistant counterparts, T-47D/TR and ZR-75/TR cells (Appendix B, Figure A2f,g, respectively). As for TamR (Figure 2b), FoxO3a is downregulated in T-47D/TR and ZR-75/TR cell lines (Appendix B, Figure A2a,b), and its overexpression significantly reduced the motility and the invasivity of transiently transfected T-47D/TR (Appendix B, Figure A2h,i). A similar trend was observed in ZR-75/TR, but FoxO3a's effect on migration and invasion (Appendix B, Figure A2j,k) was not significant, although consistent with the lower ability of FoxO3a in inducing integrin α5 expression in these cells (Appendix B, Figure A2g). Therefore, additional factors might be involved in the control of tumor progression in this particular cell line.
On the other side, in ERα− MDA-MB-231 BCCs, FoxO3a failed to induce the integrin α5 subunit (Appendix B, Figure A2l). This result is in line with data obtained through the analysis of breast cancer data sets, available on the www.cbioportal.org website (accessed on 2 October 2021, see Section 2 for searching criteria). The analysis was conducted on ERα+ or ERα− BC patients subgroups and revealed a strong correlation between FoxO3a and integrin α5 mRNA expression only in patients with ERα+ BC, while the correlation was lost in ERα− BC patients (Figure 3i). These results suggest that the FoxO3a-dependent induction of integrin α5 is not cell type-specific, but it represents a general mechanism occurring in ERα+ breast tumors only, but not in ERα− ones.

FoxO3a Binds to and Transactivates the Human Integrin alpha5 Promoter
Since FoxO3a regulation of the integrin α5 occurs not only at the protein (Figure 3c) but also at the mRNA level (Figure 3b), we questioned if the nuclear factor could transcriptionally control the integrin α5 expression. Indeed, through an accurate analysis of the human integrin α5 promoter, we identified a Forkhead core sequence (FHRE), GTTTAC between the position −329/−323 from the TSS (see Section 2 for details). As supposed, FoxO3a overexpression significantly induced the activity of a vector bearing the luciferase gene under the control of the −679/+19 region of the integrin α5 promoter (pGL3pα5) (Figure 4a,b). Interestingly, the construct (pGL3pα5-del), lacking the FHRE core sequence, failed to be induced by FoxO3a (Figure 4c). The same results were obtained in TamR/TetOn-AAA cells (Appendix C).
The involvement of FoxO3a in the transcriptional activation of the α5 promoter was corroborated by chromatin immunoprecipitation (ChIP) experiments, which evidenced significant recruitment of FoxO3a on the region containing the −329/−323 FHRE sequence (Figure 4d) at 16 h and 24 h from protein induction in TamR/TetOn-AAA cells (Figure 4e). FoxO3a recruitment was consistent with the relative amounts of the transcription factor in each sample at both time points (Figure 4e, right panel). Altogether, these data suggest that the integrin α5 subunit of the fibronectin receptor is a FoxO3a-regulated gene. representation of the human integrin α5 promoter plasmid (pGL3pα5) and the FHRE deleted fragment (pGL3pα5-del). The constructs contain the same 3′-boundary (+19), while the 5′boundaries of the promoter were −679 and −315, respectively. (b,c) Transcriptional activity of integrin α5 promoter fragments following FoxO3a overexpression is shown. TamR cells (7 × 10 4 /well) were co-transfected in suspension either with pcDNA3.1 or F3aAAA vectors (0.5 μg/well) and seeded in GM on 24-well plates. After 36 h, a second transfection with pGL3pα5 (b) or pGL3pα5-del (c) plus pRL-Tk plasmid occurred. The next day, the firefly luciferase activity was detected. Data obtained from at least three independent experiments were normalized to the activity of the pRL-Tk and expressed as relative luciferase activity (fold induction over control). FoxO3a expression in transfected samples was analyzed by WB on total lysates from the same set of cells. (d) A screenshot from NCBI genome data viewer illustrating FHRE location in the 5′ flanking region of the ITGA5 gene. FHRE motif is indicated by a red box and goes from position 54419543 to 54419550 in the negative strand. For ChIP analysis, the FHRE-containing DNA region, spanning . FoxO3a binds to and transactivates the human integrin α5 promoter. (a) Schematic representation of the human integrin α5 promoter plasmid (pGL3pα5) and the FHRE deleted fragment (pGL3pα5-del). The constructs contain the same 3 -boundary (+19), while the 5 -boundaries of the promoter were −679 and −315, respectively. (b,c) Transcriptional activity of integrin α5 promoter fragments following FoxO3a overexpression is shown. TamR cells (7 × 10 4 /well) were co-transfected in suspension either with pcDNA3.1 or F3aAAA vectors (0.5 µg/well) and seeded in GM on 24-well plates. After 36 h, a second transfection with pGL3pα5 (b) or pGL3pα5-del (c) plus pRL-Tk plasmid occurred. The next day, the firefly luciferase activity was detected. Data obtained from at least three independent experiments were normalized to the activity of the pRL-Tk and expressed as relative luciferase activity (fold induction over control). FoxO3a expression in transfected samples was analyzed by WB on total lysates from the same set of cells. (d) A screenshot from NCBI genome data viewer illustrating FHRE location in the 5 flanking region of the ITGA5 gene. FHRE motif is indicated by a red box and goes from position 54419543 to 54419550 in the negative strand. For ChIP analysis, the FHRE-containing DNA region, spanning from position 54419462 to 54419566 of the 5 flanking region of the ITGA5 gene located on chromosome 12 (e) was precipitated with ChIP-grade anti-FoxO3a antibody from nuclear extracts of TamR/TetOn-AAA cells, treated or not with Dox 1 µg/mL for 16 and 24 h. Purified chromatin was then amplified using a specific pair of primers (indicated by arrows in panel (d); see Section 2 for sequences). FoxO3a and integrin α5 expression were analyzed by WB on protein extracts from the same set of cells (right panel). Data represent the mean ± s.d. of at least three independent experiments. ** p ≤ 0.01, *** p ≤ 0.001 vs. control.

Role of Integrin a5 in FoxO3a-Dependent Inhibition of TamR Cells Migration and Invasion and Clinical Implications
Finally, to assess if integrin α5 is a mediator of the inhibitory role of FoxO3a on motility and invasiveness, silencing experiments were conducted in TamR cells by using a specific and validated siRNA, targeting integrin α5 (siα5) ( Figure 5). Interestingly, siα5 was able to counteract FoxO3a-dependent effects. Indeed, integrin α5 knock-down restored to control (pcDNA3.1-siScramble) levels the migrating ( Figure 5a) and invading (Figure 5b) potential of F3aAAA overexpressing cells, thus confirming that integrin α5 is a mediator of the FoxO3a-dependent inhibition of TamR cells motility and invasiveness. chromosome 12 (e) was precipitated with ChIP-grade anti-FoxO3a antibody from nuclear extracts of TamR/TetOn-AAA cells, treated or not with Dox 1 μg/mL for 16 and 24 h. Purified chromatin was then amplified using a specific pair of primers (indicated by arrows in panel (d); see Section 2 for sequences). FoxO3a and integrin α5 expression were analyzed by WB on protein extracts from the same set of cells (right panel). Data represent the mean ± s.d. of at least three independent experiments. ** p ≤ 0.01, *** p ≤ 0.001 vs. control.

Role of Integrin a5 in FoxO3a-Dependent Inhibition of TamR Cells Migration and Invasion and Clinical Implications
Finally, to assess if integrin α5 is a mediator of the inhibitory role of FoxO3a on motility and invasiveness, silencing experiments were conducted in TamR cells by using a specific and validated siRNA, targeting integrin α5 (siα5) ( Figure 5). Interestingly, siα5 was able to counteract FoxO3a-dependent effects. Indeed, integrin α5 knock-down restored to control (pcDNA3.1-siScramble) levels the migrating (Figure 5a) and invading (Figure 5b) potential of F3aAAA overexpressing cells, thus confirming that integrin α5 is a mediator of the FoxO3a-dependent inhibition of TamR cells motility and invasiveness.
We previously reported how high levels of FoxO3a are associated with DMFS and RFS (see Section 2) in tamoxifen-treated luminal A BC patients only [9]. An up-to-date version of those K-M curves can be found in Appendix D, which confirms that FoxO3a correlates with a better prognosis in this subset of patients. Interestingly, in the same cohort, FoxO3a and integrin α5 co-expression strongly improve DMFS (log-rank test, p = 0.0026, Figure 5c) compared to FoxO3a alone (log-rank test, p = 0.02, Appendix A4a), corroborating the hypothesis that integrin α5 could emphasize FoxO3a protective role in ERα+ BC patients. On the other hand, FoxO3a-positive prognostic value on RFS (log-rank test, p = 0.013, Appendix A4b) was not significantly affected by integrin α5 co-expression (log-rank test, p = 0.017, Figure 5d).  A schematic representation of the main findings presented here is shown in Figure 6.

Discussion
BC is the most common female malignancy worldwide [1], with ERα positivity accounting for more than 70% of all diagnoses [34] and tamoxifen still representing the mainstay of treatment and prevention for this BC subtype [35]. A 50% reduction in the risk of relapse and a 31% reduction in the annual BC death rate was reported for 5 years of tamoxifen adjuvant therapy at 15 years of follow-up [36]. Despite this success, ~20%-30% of patients develop resistance to the treatment within 3-5 years [37]. Therefore, the search for additional molecular targets that could potentially be exploited in combination with endocrine therapies continues to be an urgent clinical need. Among the multiple mechanisms that have been described, spanning from the dysregulation of ERα signaling and cell cycle modulators to the hyper-activation of GFs pathways [38], the loss of members belonging to the Forkhead class O family has been involved in the acquisition Figure 5. Role of integrin α5 in FoxO3a-dependent inhibition of TamR cells migration and invasion and clinical implications. TamR cells were silenced for integrin α5 as described in Section 2. The day after, cells were transiently transfected with F3aAAA for 24 h. pcDNA3.1 was used as a control vector. Cells were then employed for migration (a) or invasion (b) assays and counted using ImageJ software. The graphs report the % of migrating and invading cells. WB analysis was conducted on a duplicate set of cells to assess the effective depletion of integrin α5 and overexpression of FoxO3a. β-Actin was used as a loading control. Results represent the mean ± s.d. of at least three independent experiments (* p ≤ 0.05, *** p ≤ 0.001). Kaplan-Meier curve for (c) DMFS (485 patients) and (d) RFS (577 patients) were evaluated in a cohort of luminal A subtype BC patients treated with tamoxifen and co-expressing both FoxO3a and integrin a5 transcripts.
We previously reported how high levels of FoxO3a are associated with DMFS and RFS (see Section 2) in tamoxifen-treated luminal A BC patients only [9]. An up-to-date version of those K-M curves can be found in Appendix D, which confirms that FoxO3a correlates with a better prognosis in this subset of patients. Interestingly, in the same cohort, FoxO3a and integrin α5 co-expression strongly improve DMFS (log-rank test, p = 0.0026, Figure 5c) compared to FoxO3a alone (log-rank test, p = 0.02, Appendix D Figure A4a), corroborating the hypothesis that integrin α5 could emphasize FoxO3a protective role in ERα+ BC patients. On the other hand, FoxO3a-positive prognostic value on RFS (log-rank test, p = 0.013, Appendix D Figure A4b) was not significantly affected by integrin α5 co-expression (log-rank test, p = 0.017, Figure 5d).
A schematic representation of the main findings presented here is shown in Figure 6.  A schematic representation of the main findings presented here is shown in Figure 6.

Discussion
BC is the most common female malignancy worldwide [1], with ERα positivity accounting for more than 70% of all diagnoses [34] and tamoxifen still representing the mainstay of treatment and prevention for this BC subtype [35]. A 50% reduction in the risk of relapse and a 31% reduction in the annual BC death rate was reported for 5 years

Discussion
BC is the most common female malignancy worldwide [1], with ERα positivity accounting for more than 70% of all diagnoses [34] and tamoxifen still representing the mainstay of treatment and prevention for this BC subtype [35]. A 50% reduction in the risk of relapse and a 31% reduction in the annual BC death rate was reported for 5 years of tamoxifen adjuvant therapy at 15 years of follow-up [36]. Despite this success,~20%-30% of patients develop resistance to the treatment within 3-5 years [37]. Therefore, the search for additional molecular targets that could potentially be exploited in combination with endocrine therapies continues to be an urgent clinical need. Among the multiple mechanisms that have been described, spanning from the dysregulation of ERα signaling and cell cycle modulators to the hyper-activation of GFs pathways [38], the loss of members belonging to the Forkhead class O family has been involved in the acquisition of tamoxifen resistance. In particular, the pro-apoptotic Forkhead transcription factor FoxO1 (formerly FKHR) failed to be activated in response to tamoxifen in a TamR MCF-7 BCC model [39]. Accordingly, Vaziri-Gohar et al. observed a decreased expression of FoxO1 in MCF-7/TamR cells and reduced sensitivity to tamoxifen in FoxO1-deficient parental MCF-7 [40]. Notably, FoxO1 is a FoxO3a target gene [41]. Since we recently reported that FoxO3a is downregulated in TamR BCC models and that its induction is able to restore the sensitivity to tamoxifen both in vitro and in vivo [9], very likely that the above described FoxO1 reduction in TamR cells is a consequence of FoxO3a hyper-phosphorylation and degradation in these cells [9]. On the other hand, being the potent oncogene FoxM1, another member of the Forkhead family, negatively regulated by FoxO3a [42], its increased expression in more invasive and endocrine-resistant tumors [43,44], perfectly fits with the evidence of a defective FoxO3a in this subset of cancers.
Several other findings have demonstrated that integrins, transmembrane receptors involved in cell adhesion to ECM, are important regulators of tumor initiation and progression, cancer stemness, and drug resistance, but the available data are often conflicting [45]. For instance, subunits α6 [46] and β1 [47,48] have been positively associated with tamoxifen resistance and with tumor progression [31,49]. Nevertheless, the expression of several integrins has been found reduced in BC specimens compared with benign tumors, and it was inversely related to the presence of axillary metastasis [50]. Accordingly, the expression of integrin α5 subunit was high in non-metastatic BCCs and low in invasive cell lines, where integrin α5 overexpression was able to inhibit cell proliferation, migration, and invasion [31]. Moreover, we reported that 17β-estradiol-activated ERα increases integrin α5 expression in ERα expressing BCCs [27], suggesting that integrin α5 subunit might function as a potential metastasis suppressor, at least in ERα+ tumors.
Of note, clinical trials conducted on αVβ3/αVβ5 and α5β1 integrin inhibitors have globally failed to demonstrate therapeutic benefits in blocking tumor growth and progression, and the results were either inconsistent or even favoring placebo (!) [51].
The role of the integrin α5 subunit in antiestrogen-resistant BC has never been addressed. Here we show that, aside from FoxO3a [9], also the α5 (but not the β1) subunit of the integrin α5β1 fibronectin receptor is downregulated in TamR compared to MCF-7 parental cells. Oddly, our data are in contrast with previous observations reporting an increased β1 in a similar MCF-7-derived tamoxifen-resistant (MCF-7R) cell model, but it has to be underlined that cells were grown in quite different culture conditions [48].
We also show that FoxO3a re-expression or activation by means of PI3K/AKT pathway inhibitors induce the α5, but not the β1, integrin subunit, both in vitro and in vivo. The integrin α5 regulation likely occurs at the transcriptional level since FoxO3a is recruited to an FHRE motif identified on the ITGA5 gene promoter. The evidence of a decreased integrin α5 expression in FoxO3a silenced cells confirmed that integrin α5 is regulated by FoxO3a.
FoxO3a-dependent increase in integrin α5 is accompanied, in turn, by a significant reduction in TamR cell migration and invasion. Interestingly, the effect was only observed in ERα+ BCCs, while integrin α5 protein levels remained unchanged in FoxO3a overex-pressing ERα− cells. This evidence corroborates our previously published results, showing that ligand-activated ERα induces integrin α5 expression in ERα+ BCCs, reducing their migrating and invading potential, thus demonstrating that integrin α5 expression contributes to the maintenance of a stationary status in ERα+ cells [27]. This assumption was strongly supported by clinical data from large-scale gene expression data sets (TCGA database), which revealed a highly significant correlation between FOXO3 and ITGA5 expression in ERα+, but not in ERα−, BC patients (Figure 3i). Moreover, K-M analysis conducted on a tamoxifen-treated luminal A BC cohort of patients, with long-term follow-up (180 months), showed an even increased protection against recurrence and distant metastasis when both FoxO3a and integrin α5 are co-expressed (Figure 5c,d) compared to FoxO3a alone (Appendix D). Therefore, low levels of FoxO3a in association with a decrease in integrin α5 expression could identify high-risk luminal A subtype BC patients.
Altogether, our data show that the induction of integrin α5 expression might represent one of the mechanisms through which FoxO3a restores the tumor response to antiestrogen treatment, confirming that the transcription factor might represent a pursuable target to be exploited in endocrine-resistant tumors.

Conclusions
The hyper-activation of the GFs pathway is one of the most important features leading to antiestrogen resistance. Therefore, several drugs targeting the PI3K/AKT/mTOR axis are currently being investigated in clinical trials in combination with standard therapies to overcome acquired resistance to common BC treatments, including tamoxifen [6]. Despite the dysregulation of FoxOs factors has emerged as a key molecular feature of endocrine resistance mechanisms [9,24,52,53], very few clinical trials investigating the efficacy of PI3K or AKT inhibitors in BC tumors, included the evaluation of FoxO3a expression and/or subcellular localization as a secondary outcome (https://clinicaltrials.gov/, Identifiers: NCT02077569 [54], NCT02260661, NCT01339442, accessed on 10 September 2021). Among these studies, only the STAKT study, conducted on the oral selective pan-AKT inhibitor capivasertib (AZD5363), has results showing an increase in p-AKT content and FoxO3a nuclear translocation (thus activation) following treatment, which are both consistent with an inhibition of the AKT pathway [54]. However, cancer cells can employ a multitude of different mechanisms to acquire resistance to PI3K-AKT inhibition [24]. To overcome this inconvenience, restoration of FoxO3a expression and/or activity [26,55], which translates in the regulation of FoxO3a multiple transcriptional targets, including integrin α5, could represent a promising and reliable approach to be exploited as an adjuvant to antiestrogen therapy in stratified BC patients that are refractory to the treatment.    (10 6 ) were seeded in GM on agar stratified 100 mm dishes, treated with Dox 1 µg/mL, and monitored for up to 72 h. Dox was renewed after 48 h. Images were taken at 10× magnification. The graph represents the mean of spheroids number and size. (i) Confluent cells were subjected to wound-healing scratch assay and treated with Dox 1 µg/mL. Dox-induced FoxO3a (TamR/TetOn-AAA) significantly hinders wound closure at all time points compared to the control cell line (TamR/TetOn-V). Proteins were collected from a duplicate set of cells and subjected to WB analysis to assess the efficiency of FoxO3a induction (right panel). Cells were seeded, treated with Dox 1 µg/mL for 48 h, and then harvested with Versene and subjected to migration (j) and invasion (k) assays. All graphs represent the mean ± s.d. of at least three independent experiments (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

Appendix B
Cancers 2022, 14, x FOR PEER REVIEW 19 of 23  (c) Total proteins were extracted from TamR/TetOn-AAA and from TamR/TetOn-V control-derived tumors, previously explanted from Dox-treated mice [9], and subjected to WB analysis to evaluate β1 expression (β-Actin: loading control). Dox-induced FoxO3a (for 48 h) increased integrin α5 mRNA (d) and protein (e) content in TamR/TetOn-AAA cells but not in control TamR/TetOn-V cells. A higher protein expression of integrin α5 was observed in the other two FoxO3a overexpressing, ERα+ BC cell lines, (f) T-47D and (g) ZR-75 as well as in their tamoxifenresistant counterparts. FoxO3a ectopic expression also affected T-47D (h,i) and, in a lesser extent, ZR-75 (j,k) motility and invasiveness. All the experiments conducted on T-47D, ZR-75, and their Tam-resistant cell lines followed the same experimental procedure already described for MCF-7 and TamR cells. (l) FoxO3a overexpression does not induce integrin α5 in ERα− BCCs: TNBC MDA-MB-231 were transiently transfected in suspension, in PRF-GM, with 3 µg/60 mm dish of F3aAAA or pcDNA3.1. After 48 h, total lysates were collected, and FoxO3a and integrin α5 content was assessed through WB analysis. β-actin was used as a loading control. Results represent the mean ± s.d. of at least three independent experiments (** p ≤ 0.01). Figure A3. FoxO3a transactivates the human integrin α5 promoter. TamR/TetOn-AAA cells were seeded on 24-well plates in GM plus Dox 1 μg/mL. After 36 h, they were co-transfected either with (a) pGL3pα5 or (b) pGL3pα5-del and pRL-Tk. The next day cells were lysed and subjected to luciferase assay. FoxO3a expression in transfected samples was analyzed by WB on total lysates from the same set of cells. Data obtained from at least three independent experiments were normalized to the activity of the pRL-Tk and expressed as fold induction over control (** p ≤ 0.01). Figure A4. FoxOa is a positive prognostic factor in luminal A BC patients. Distant metastasis-free survival (DMFS) and relapse-free survival (RFS) were evaluated in a cohort of luminal A subtype patients treated with tamoxifen (Endoc. Ther.: Tam) (DMFS-485 patients (a), RFS-577 patients (b)). K-M analysis was performed regardless of specific chemotherapeutic treatments (Chemoth.: any). K-M are plotted for high (above median, in red) and low (below median, in black) FoxO3a gene expression. Search parameters are reported in Section 2. p-values were calculated using the log-rank test and plotted in R.